Limbic system influences on motor behavior seem widespread, and could range from the initiation of action to the motivational pace of motor output. Motor abnormalities are also a common feature of psychiatric illness. Several subcortical limbic-motor entry points have been defined in recent years, but cortical entry points are understood poorly, despite the fact that a part of the limbic lobe, the cingulate motor cortex (area 24c or M3, and area 23c or M4), contributes axons to the corticospinal pathway. Using retrograde and anterograde tracers in rhesus monkeys, we investigated the ipsilateral limbic input to area 24c and adjacent area 23c. Limbic cortical input to areas 24c and 23c arise from cingulate areas 24a, 24b, 23a, 23b, and 32, retrosplenial areas 30 and 29, and temporal areas 35, TF and TH. Areas 24c and 23c were also interconnected strongly. The dysgranular part of the orbitofrontal cortex and insula projects primarily to area 24c while the granular part of the orbitofrontal cortex and insula projects primarily to area 23c. Afferents from cingulate area 25, the retrocalcarine cortex, temporal pole, entorhinal cortex, parasubiculum, and the medial part of area TH target primarily or only area 24c. Our findings indicate that a variety of telencephalic limbic afferents converge on cortex lining the lower bank and fundus of the anterior part of the cingulate sulcus. Because it is known that this cortex gives rise to axons ending in the spinal cord, facial nucleus, pontine gray, red nucleus, putamen, and primary and supplementary motor cortices, we suggest that the cingulate motor cortex forms a strategic cortical entry point for limbic influence on the voluntary motor system.

BACKGROUND

Protease-activated receptor-1 (PAR-1) is the high-affinity receptor for the coagulation protease thrombin. It is expressed by a variety of cell types in the heart, including cardiomyocytes and cardiac fibroblasts. We have shown that tissue factor (TF) and thrombin contribute to infarct size after cardiac ischemia-reperfusion (I/R) injury. Moreover, in vitro studies have shown that PAR-1 signaling induces hypertrophy of cardiomyocytes and proliferation of cardiac fibroblasts. The purpose of the present study was to investigate the role of PAR-1 in infarction, cardiac remodeling, and hypertrophy after I/R injury. In addition, we analyzed the effect of overexpression of PAR-1 on cardiomyocytes.

RESULTS

We found that PAR-1 deficiency reduced dilation of the left ventricle and reduced impairment of left ventricular function 2 weeks after I/R injury. Activation of ERK1/2 was increased in injured PAR-1(-/-) mice compared with wild-type mice; however, PAR-1 deficiency did not affect infarct size. Cardiomyocyte-specific overexpression of PAR-1 in mice induced eccentric hypertrophy (increased left ventricular dimension and normal left ventricular wall thickness) and dilated cardiomyopathy. Deletion of the TF gene in cardiomyocytes reduced the eccentric hypertrophy in mice overexpressing PAR-1.

CONCLUSIONS

Our results demonstrate that PAR-1 contributes to cardiac remodeling and hypertrophy. Moreover, overexpression of PAR-1 on cardiomyocytes induced eccentric hypertrophy. Inhibition of PAR-1 after myocardial infarction may represent a novel therapy to reduce hypertrophy and heart failure in humans.

Transcriptional networks, which are initiated by secreted proteins, cooperate with each other to orchestrate eye development. The establishment of dorsal/ventral polarity, especially dorsal specification in the optic vesicle, is poorly understood at a molecular and cellular level. Here, we show that COUP-TFI (Nr2f1) and COUP-TFII (Nr2f2) are highly expressed in the progenitor cells in the developing murine eye. Phenotype analysis of COUP-TFI and COUP-TFII single-gene conditional knockout mouse models suggests that COUP-TFs compensate for each other to maintain morphogenesis of the eye. However, in eye-specific COUP-TFI/TFII double-knockout mice, progenitor cells at the dorso-distal optic vesicle fail to differentiate appropriately, causing the retinal pigmented epithelium cells to adopt a neural retina fate and abnormal differentiation of the dorsal optic stalk; the development of proximo-ventral identities, neural retina and ventral optic stalk is also compromised. These cellular defects in turn lead to congenital ocular colobomata and microphthalmia. Immunohistochemical and in situ hybridization assays reveal that the expression of several regulatory genes essential for early optic vesicle development, including Pax6, Otx2, Mitf, Pax2 and Vax1/2, is altered in the corresponding compartments of the mutant eye. Using ChIP assay, siRNA treatment and transient transfection in ARPE-19 cells in vitro, we demonstrate that Pax6 and Otx2 are directly regulated by COUP-TFs. Taken together, our findings reveal novel and distinct cell-intrinsic mechanisms mediated by COUP-TF genes to direct the specification and differentiation of progenitor cells, and that COUP-TFs are crucial for dorsalization of the eye.

Tea is the most popular beverage, consumed by over two thirds of the world's population. Tea is processed differently in different parts of the world to give green (20%), black (78%) or oolong tea (2%). Green tea is consumed mostly in Japan and China. The antimutagenic and anticarcinogenic activities of green tea are extensively examined. The chemical components of green and black tea are polyphenols, which include EC, ECG, EGC, EGCG and TFs. This article reviews the epidemiological and experimental studies on the antimutagenicity and anticarcinogenicity of tea extracts and tea polyphenols. In Japan, an epidemiological study showed an inverse relationship between habitual green tea drinking and the standardized mortality rates for cancer. Some cohort studies on Chanoyu (Japanese tea ceremony) women teachers also showed that their mortality ratio including deaths caused by malignant neoplasms were surprisingly low. The antimutagenic activity against various mutagens of tea extracts and polyphenols including ECG and EGCG has been demonstrated in microbial systems (Salmonella typhimurium and Escherichia coli), mammalian cell systems and in vivo animal tests. The anticarcinogenic activity of tea phenols has been shown in experimental animals such as rats and mice, in transplantable tumors, carcinogen-induced tumors in digestive organs, mammary glands, hepatocarcinomas, lung cancers, skin tumors, leukemia, tumor promotion and metastasis. The mechanisms of antimutagenesis and anticarcinogenesis of tea polyphenols suggest that the inhibition of tumors may be due to both extracellular and intracellular mechanisms including the modulation of metabolism, blocking or suppression, modulation of DNA replication and repair effects, promotion, inhibition of invasion and metastasis, and induction of novel mechanisms.

The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around -120 to -80 bp, while highly effective sgRNAs targeted from -147 to -89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4. Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells.

OBJECTIVE

To quantify fundus autofluorescence (qAF) in patients with recessive Stargardt disease (STGD1).

METHODS

A total of 42 STGD1 patients (ages: 7-52 years) with at least one confirmed disease-associated ABCA4 mutation were studied. Fundus AF images (488-nm excitation) were acquired with a confocal scanning laser ophthalmoscope equipped with an internal fluorescent reference to account for variable laser power and detector sensitivity. The gray levels (GLs) of each image were calibrated to the reference, zero GL, magnification, and normative optical media density to yield qAF. Texture factor (TF) was calculated to characterize inhomogeneities in the AF image and patients were assigned to the phenotypes of Fishman I through III.

RESULTS

Quantified fundus autofluorescence in 36 of 42 patients and TF in 27 of 42 patients were above normal limits for age. Young patients exhibited the relatively highest qAF, with levels up to 8-fold higher than healthy eyes. Quantified fundus autofluorescence and TF were higher in Fishman II and III than Fishman I, who had higher qAF and TF than healthy eyes. Patients carrying the G1916E mutation had lower qAF and TF than most other patients, even in the presence of a second allele associated with severe disease.

CONCLUSIONS

Quantified fundus autofluorescence is an indirect approach to measuring RPE lipofuscin in vivo. We report that ABCA4 mutations cause significantly elevated qAF, consistent with previous reports indicating that increased RPE lipofuscin is a hallmark of STGD1. Even when qualitative differences in fundus AF images are not evident, qAF can elucidate phenotypic variation. Quantified fundus autofluorescence will serve to establish genotype-phenotype correlations and as an outcome measure in clinical trials.

Trigger factor (TF) in Escherichia coli is a molecular chaperone with remarkable properties: it has prolyl-isomerase activity, associates with nascent polypeptides on ribosomes, binds to GroEL, enhances GroEL's affinity for unfolded proteins, and promotes degradation of certain polypeptides. Because the latter effects appeared larger at 20 degrees C, we studied the influence of temperature on TF expression. Unlike most chaperones (e.g., GroEL), which are heat-shock proteins (hsps), TF levels increased progressively as growth temperature decreased from 42 degrees C to 16 degrees C and even rose in cells stored at 4 degrees C. Upon temperature downshift from 37 degrees C to 10 degrees C or exposure to chloramphenicol, TF synthesis was induced, like that of many cold-shock proteins. We therefore tested if TF expression might be important for viability at low temperatures. When stored at 4 degrees C, E. coli lose viability at exponential rates. Cells with reduced TF content die faster, while cells overexpressing TF showed greater viability. Although TF overproduction protected against cold, it reduced viability at 50 degrees C, while TF deficiency enhanced viability at this temperature. By contrast, overproduction of GroEL/ES, or hsps generally, while protective against high temperatures, reduced viability at 4 degrees C, which may explain why expression of hsps is suppressed in the cold. Thus, TF represents an example of an E. coli protein which protects cells against low temperatures. Moreover, the differential induction of TF at low temperatures and hsps at high temperatures appears to provide selective protection against these opposite thermal extremes.

BACKGROUND

Amhara Regional State of Ethiopia has a population of approximately 19.6 million, is prone to unstable and epidemic malaria, and is severely affected by trachoma. An integrated malaria and trachoma control program is being implemented by the Regional Health Bureau. To provide baseline data, a survey was conducted during December 2006 to estimate malaria parasite prevalence, malaria indicators, prevalence of trachoma, and trachoma risk factors in households and people of all ages in each of the ten zones of the state, excluding three urban centers (0.4% of the population).

RESULTS

The study was designed to provide prevalence estimates at zone and state levels. Using multi-stage cluster random sampling, 16 clusters of 25 households were randomly selected in each of the ten zones. Household heads were interviewed for malaria indicators and trachoma risk factors (N = 4,101). All people were examined for trachoma signs (N = 17,242), and those in even-numbered households provided blood films for malaria parasite detection (N = 7,745); both thick and thin blood films were read. Zonal malaria parasite prevalence ranged from 2.4% to 6.1%, with the overall state-wide prevalence being 4.6% (95% confidence interval (CI): 3.8%-5.6%). The Plasmodium falciparum: Plasmodium vivax ratio ranged from 0.9-2.1 with an overall regional ratio of 1.2. A total of 14.8% of households reported indoor residual spraying in the past year, 34.7% had at least one mosquito net, and 16.1% had one or more long-lasting insecticidal net. Zonal trachoma prevalence (trachomatous inflammation follicular [WHO grade TF] in children aged 1-9 years) ranged from 12.6% to 60.1%, with the overall state-wide prevalence being 32.7% (95% CI: 29.2%-36.5%). State-wide prevalence of trachomatous trichiasis (TT) in persons aged over fifteen was 6.2% (95% CI: 5.3-7.4), and 0.3% (95% CI: 0.2-0.5) in children aged 0-14 years. Overall, an estimated 643,904 persons (lower bound 419,274, upper bound 975,635) have TT and require immediate corrective surgery.

CONCLUSIONS

The results provide extensive baseline data to guide planning, implementation, and evaluation of the integrated malaria and trachoma control program in Amhara. The success of the integrated survey is the first step towards demonstration that control of priority neglected tropical diseases can be integrated with one of the "big three" killer diseases.

The core embryonic stem cell transcription factors (TFs) OCT3/4 (OCT4), NANOG, and SOX2 have shared as well as nonoverlapping roles in stem cell growth and differentiation. These same TFs are also expressed in various types of human germ cell tumors (GCTs), implicating them in regulation of tumor growth and differentiation. Although NANOG and OCT3/4 are sensitive and specific markers for seminoma and embryonal carcinoma, neither factor aids in the clinically important distinction of seminomatous from nonseminomatous tumors. In contrast, expression profiling data suggest that SOX2 may help with this distinction. To determine if a panel of embryonic stem cell TFs (NANOG, OCT3/4, and SOX2) can facilitate the identification and distinction of seminomatous from nonseminomatous GCTs, we evaluated their expression by immunohistochemistry in primary testicular (n=41) and metastatic retroperitoneal (n=43) GCTs. Our results confirm NANOG and OCT3/4 as sensitive and specific markers for primary seminoma and embryonal carcinoma and demonstrate the novel finding that NANOG is a marker for metastatic GCTs. In addition, SOX2 is expressed in embryonal carcinoma but not pure seminoma and is therefore a useful diagnostic marker for distinguishing seminomatous and nonseminomatous GCTs. In summary, we find that the embryonic stem cell TF signature of seminoma is NANOG+, OCT3/4+, and SOX2-, whereas embryonal carcinoma is NANOG+, OCT3/4+, and SOX2+, and expect these immunohistochemical profiles will facilitate the diagnosis of both primary and metastatic GCTs.

The subcellular location of radiolabeled transferrin (125I-Tf), internalized during cellular iron uptake, and the cellular distribution of transferrin (Tf) receptors were studied in cultured HeLa cells. Cells were incubated at 37 degrees C with 125I-Tf(Fe)2. Forty per cent of the labeled ligand was associated with cell surface receptors. The remaining 60% was internalized as shown by the inability to dissociate 125I-Tf from cells by competition with excess Tf(Fe)2 or treatment of cells with 0.2 M acetic acid containing 0.5 M NaCl. Subcellular fractionation studies using sucrose density gradients indicated that internalized Tf was localized in a membranous vesicle distinct from lysosomes, Golgi apparatus, endoplasmic reticulum, or plasma membranes. The subcellular distribution of Tf receptors was studied using an assay for detergent solubilized receptors. Even without preincubation with ligand, the majority of cellular Tf receptors were localized intracellularly in a vesicle with the same buoyant density as the vesicle containing internalized 125I-Tf. Using an assay for occupied receptors, we demonstrated that the same vesicle contained both internal receptors and internalized ligand. A portion (20%) of the intracellular receptor pool was insensitive to trypsin treatment of whole cells at 37 degrees C suggesting that during the experimental time period (20-30 min) this portion did not recycle to the cell surface. We propose that during cellular iron uptake, Tf receptor-ligand complexes are internalized and directed to a nonlysosomal compartment where iron is released, followed by recycling to the cell surface of an intact Tf receptor-apo-Tf complex.

The stress-activated kinase p38 plays key roles in tumor suppression and induction of tumor cell dormancy. However, the mechanisms behind these functions remain poorly understood. Using computational tools, we identified a transcription factor (TF) network regulated by p38alpha/beta and required for human squamous carcinoma cell quiescence in vivo. We found that p38 transcriptionally regulates a core network of 46 genes that includes 16 TFs. Activation of p38 induced the expression of the TFs p53 and BHLHB3, while inhibiting c-Jun and FoxM1 expression. Furthermore, induction of p53 by p38 was dependent on c-Jun down-regulation. Accordingly, RNAi down-regulation of BHLHB3 or p53 interrupted tumor cell quiescence, while down-regulation of c-Jun or FoxM1 or overexpression of BHLHB3 in malignant cells mimicked the onset of quiescence. Our results identify components of the regulatory mechanisms driving p38-induced cancer cell quiescence. These may regulate dormancy of residual disease that usually precedes the onset of metastasis in many cancers.

BACKGROUND

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor (TF) that mediates responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Integration of TCDD-induced genome-wide AhR enrichment, differential gene expression and computational dioxin response element (DRE) analyses further elucidate the hepatic AhR regulatory network.

RESULTS

Global ChIP-chip and gene expression analyses were performed on hepatic tissue from immature ovariectomized mice orally gavaged with 30 μg/kg TCDD. ChIP-chip analysis identified 14,446 and 974 AhR enriched regions (1% false discovery rate) at 2 and 24 hrs, respectively. Enrichment density was greatest in the proximal promoter, and more specifically, within ± 1.5 kb of a transcriptional start site (TSS). AhR enrichment also occurred distal to a TSS (e.g. intergenic DNA and 3' UTR), extending the potential gene expression regulatory roles of the AhR. Although TF binding site analyses identified over-represented DRE sequences within enriched regions, approximately 50% of all AhR enriched regions lacked a DRE core (5'-GCGTG-3'). Microarray analysis identified 1,896 number of TCDD-responsive genes (|fold change| ≥ 1.5, P1(t)>> 0.999). Integrating this gene expression data with our ChIP-chip and DRE analyses only identified 625 differentially expressed genes that involved an AhR interaction at a DRE. Functional annotation analysis of differentially regulated genes associated with AhR enrichment identified overrepresented processes related to fatty acid and lipid metabolism and transport, and xenobiotic metabolism, which are consistent with TCDD-elicited steatosis in the mouse liver.

CONCLUSIONS

Details of the AhR regulatory network have been expanded to include AhR-DNA interactions within intragenic and intergenic genomic regions. Moreover, the AhR can interact with DNA independent of a DRE core suggesting there are alternative mechanisms of AhR-mediated gene regulation.

BACKGROUND

Blood-borne tissue factor (TF) plays a crucial role in thrombogenesis.

OBJECTIVE

To study whether polymorphonuclear leukocytes (PMN) are a source of TF.

RESULTS

Human PMN were carefully separated from other blood cells and stimulated for 3 min with purified P-selectin or the chemotactic peptide formyl-MetLeuPhe (fMLP): they expressed both TF procoagulant activity, as identified by specific TF MoAb and inactivated factor VIIa blockade; and TF:Ag (four to six times), as shown by flow-cytometry and immunocytochemistry. About 40% of permeabilized PMN, both resting and stimulated, contained TF:Ag, indicating that stimulation only modifies the location of TF:Ag within PMN. By real time-polymerase chain reaction (RT-PCR), a very low amount of TF mRNA was detectable in resting PMN, but a 3- to 5-fold increase was observed after 1-h stimulation with P-selectin or fMLP, respectively.

CONCLUSIONS

These findings suggest that TF is not constitutively expressed in peripheral PMN, but can be up-regulated and produced upon stimulation and specific gene transcription, as for instance during contact with activated platelets or endothelium. The stored TF is rapidly expressed in vitro as a functional molecule on the surface of activated PMN. The availability of PMN TF supports the relevance of inflammatory cells and their interaction with platelets for fibrin deposition and thrombus formation.

Transcription factors (TFs) are commonly deregulated in the pathogenesis of human cancer and are a major class of cancer cell dependencies. Consequently, targeting of TFs can be highly effective in treating particular malignancies, as highlighted by the clinical efficacy of agents that target nuclear hormone receptors. In this review we discuss recent advances in our understanding of TFs as drug targets in oncology, with an emphasis on the emerging chemical approaches to modulate TF function. The remarkable diversity and potency of TFs as drivers of cell transformation justifies a continued pursuit of TFs as therapeutic targets for drug discovery.

Immediate-early genes (IEGs) can be activated and transcribed within minutes after stimulation, without the need for de novo protein synthesis, and they are stimulated in response to both cell-extrinsic and cell-intrinsic signals. Extracellular signals are transduced from the cell surface, through receptors activating a chain of proteins in the cell, in particular extracellular-signal-regulated kinases (ERKs), mitogen-activated protein kinases (MAPKs) and members of the RhoA-actin pathway. These communicate through a signaling cascade by adding phosphate groups to neighboring proteins, and this will eventually activate and translocate TFs to the nucleus and thereby induce gene expression. The gene activation also involves proximal and distal enhancers that interact with promoters to simulate gene expression. The immediate-early genes have essential biological roles, in particular in stress response, like the immune system, and in differentiation. Therefore they also have important roles in various diseases, including cancer development. In this paper we summarize some recent advances on key aspects of the activation and regulation of immediate-early genes.

Gaining insights on gene regulation from large-scale functional data sets is a grand challenge in systems biology. In this article, we develop and apply methods for transcriptional regulatory network inference from diverse functional genomics data sets and demonstrate their value for gene function and gene expression prediction. We formulate the network inference problem in a machine-learning framework and use both supervised and unsupervised methods to predict regulatory edges by integrating transcription factor (TF) binding, evolutionarily conserved sequence motifs, gene expression, and chromatin modification data sets as input features. Applying these methods to Drosophila melanogaster, we predict ∼300,000 regulatory edges in a network of ∼600 TFs and 12,000 target genes. We validate our predictions using known regulatory interactions, gene functional annotations, tissue-specific expression, protein-protein interactions, and three-dimensional maps of chromosome conformation. We use the inferred network to identify putative functions for hundreds of previously uncharacterized genes, including many in nervous system development, which are independently confirmed based on their tissue-specific expression patterns. Last, we use the regulatory network to predict target gene expression levels as a function of TF expression, and find significantly higher predictive power for integrative networks than for motif or ChIP-based networks. Our work reveals the complementarity between physical evidence of regulatory interactions (TF binding, motif conservation) and functional evidence (coordinated expression or chromatin patterns) and demonstrates the power of data integration for network inference and studies of gene regulation at the systems level.

Transcription factors (TFs) and microRNAs (miRNAs) can jointly regulate target gene expression in the forms of feed-forward loops (FFLs) or feedback loops (FBLs). These regulatory loops serve as important motifs in gene regulatory networks and play critical roles in multiple biological processes and different diseases. Major progress has been made in bioinformatics and experimental study for the TF and miRNA co-regulation in recent years. To further speed up its identification and functional study, it is indispensable to make a comprehensive review. In this article, we summarize the types of FFLs and FBLs and their identified methods. Then, we review the behaviors and functions for the experimentally identified loops according to biological processes and diseases. Future improvements and challenges are also discussed, which includes more powerful bioinformatics approaches and high-throughput technologies in TF and miRNA target prediction, and the integration of networks of multiple levels.

OBJECTIVE

The aim of this study was to determine the role of AMP-activated protein kinase (AMPK) and its link to protein kinase C (PKC) in the late phase of cardioprotection afforded by ischemic preconditioning (PC) against myocardial stunning.

RESULTS

Rabbits were instrumented with a balloon occluder around a coronary artery and with a Doppler sensor to monitor the thickening fraction (TF). Conscious rabbits underwent five cycles of 5-min ischemia/5-min reperfusion (I/R) on 2 consecutive days (days 1 and 2). Reduction of TF after I/R was significantly less and recovery of TF was faster on day 2, indicating a late PC effect. PC provoked translocation of PKC- from the cytosol to the membrane and significantly increased AMPK activity by 100% immediately after PC. The mRNA level of GLUT4, a glucose transporter, was elevated by 150% at 3 h after PC, and the total protein level of GLUT4 was increased by 107% at 24 h after PC. The level of sarcolemmal GLUT4 protein after I/R on day 2 was 41% higher than its level after I/R on day 1. AMPK activation and up-regulation of GLUT4 by PC were abrogated by pre-treatment with PKC inhibitors.

CONCLUSIONS

PC activated AMPK and up-regulated GLUT4 expression in a PKC-dependent manner. This GLUT4 up-regulation at 24 h after PC may contribute to attenuation of myocardial stunning.

Tandem repeats of DNA that contain transcription factor (TF) binding sites could serve as decoys, competitively binding to TFs and affecting target gene expression. Using a synthetic system in budding yeast, we demonstrate that repeated decoy sites inhibit gene expression by sequestering a transcriptional activator and converting the graded dose-response of target promoters to a sharper, sigmoidal-like response. On the basis of both modeling and chromatin immunoprecipitation measurements, we attribute the altered response to TF binding decoy sites more tightly than promoter binding sites. Tight TF binding to arrays of contiguous repeated decoy sites only occurs when the arrays are mostly unoccupied. Finally, we show that the altered sigmoidal-like response can convert the graded response of a transcriptional positive-feedback loop to a bimodal response. Together, these results show how changing numbers of repeated TF binding sites lead to qualitative changes in behavior and raise new questions about the stability of TF/promoter binding.

NF-Y, a trimeric transcription factor (TF) composed of two histone-like subunits (NF-YB and NF-YC) and a sequence-specific subunit (NF-YA), binds to the CCAAT motif, a common promoter element. Genome-wide mapping reveals 5000-15,000 NF-Y binding sites depending on the cell type, with the NF-YA and NF-YB subunits binding asymmetrically with respect to the CCAAT motif. Despite being characterized as a proximal promoter TF, only 25% of NF-Y sites map to promoters. A comparable number of NF-Y sites are located at enhancers, many of which are tissue specific, and nearly half of the NF-Y sites are in select subclasses of HERV LTR repeats. Unlike most TFs, NF-Y can access its target DNA motif in inactive (nonmodified) or polycomb-repressed chromatin domains. Unexpectedly, NF-Y extensively colocalizes with FOS in all genomic contexts, and this often occurs in the absence of JUN and the AP-1 motif. NF-Y also coassociates with a select cluster of growth-controlling and oncogenic TFs, consistent with the abundance of CCAAT motifs in the promoters of genes overexpressed in cancer. Interestingly, NF-Y and several growth-controlling TFs bind in a stereo-specific manner, suggesting a mechanism for cooperative action at promoters and enhancers. Our results indicate that NF-Y is not merely a commonly used proximal promoter TF, but rather performs a more diverse set of biological functions, many of which are likely to involve coassociation with FOS.

Regular physical activity (PA) is increasingly promoted for people with rheumatic and musculoskeletal diseases as well as the general population. We evaluated if the public health recommendations for PA are applicable for people with inflammatory arthritis (iA; Rheumatoid Arthritis and Spondyloarthritis) and osteoarthritis (hip/knee OA) in order to develop evidence-based recommendations for advice and guidance on PA in clinical practice. The EULAR standardised operating procedures for the development of recommendations were followed. A task force (TF) (including rheumatologists, other medical specialists and physicians, health professionals, patient-representatives, methodologists) from 16 countries met twice. In the first TF meeting, 13 research questions to support a systematic literature review (SLR) were identified and defined. In the second meeting, the SLR evidence was presented and discussed before the recommendations, research agenda and education agenda were formulated. The TF developed and agreed on four overarching principles and 10 recommendations for PA in people with iA and OA. The mean level of agreement between the TF members ranged between 9.8 and 8.8. Given the evidence for its effectiveness, feasibility and safety, PA is advocated as integral part of standard care throughout the course of these diseases. Finally, the TF agreed on related research and education agendas. Evidence and expert opinion inform these recommendations to provide guidance in the development, conduct and evaluation of PA-interventions and promotion in people with iA and OA. It is advised that these recommendations should be implemented considering individual needs and national health systems.

Mycobacterium tuberculosis (MTB) infects 30% of all humans and kills someone every 20-30 s. Here we report genome-wide binding for ~80% of all predicted MTB transcription factors (TFs), and assayed global expression following induction of each TF. The MTB DNA-binding network consists of ~16,000 binding events from 154 TFs. We identify >50 TF-DNA consensus motifs and >1,150 promoter-binding events directly associated with proximal gene regulation. An additional ~4,200 binding events are in promoter windows and represent strong candidates for direct transcriptional regulation under appropriate environmental conditions. However, we also identify >10,000 'dormant' DNA-binding events that cannot be linked directly with proximal transcriptional control, suggesting that widespread DNA binding may be a common feature that should be considered when developing global models of coordinated gene expression.

Titanium implants have been used widely and successfully for various types of bone-anchored reconstructions. It is believed that properties of oxide films covering titanium implant surfaces are of crucial importance for a successful osseointegration, in particular at compromized bone sites. The aim of the present study is to investigate the surface properties of anodic oxides formed on commercially pure (c.p.) titanium screw implants as well as to study 'native' oxides on turned c.p. titanium implants. Anodic oxides were prepared by galvanostatic mode in CH3COOH up to the high forming voltage of dielectric breakdown and spark formation. The oxide thicknesses, measured with Auger electron spectroscopy (AES), were in the range of about 200-1000 nm. Barrier and porous structures dominated the surface morphology of the anodic film. Quantitative morphometric analyses of the micropore structures were performed using an image analysis system on scanning electron microscopy (SEM) negatives. The pore sizes were < or = 8 microm in diameter and had 1.27-2.1 microm2 opening area. The porosity was in the range of 12.7-24.4%. The surface roughness was in the range of 0.96-1.03 microm (Sa), measured with TopScan 3D. The crystal structures of the titanium oxide were amorphous, anatase, and a mixtures of anatase and rutile type, as analyzed with thin-film X-ray diffractometry (TF-XRD) and Raman spectroscopy. The chemical compositions consisted mainly of TiO2, characterized with X-ray photoelectron spectroscopy (XPS). The native (thermal) oxide on turned implants was 17.4 nm (+/- 6.2) thick and amorphous. Its chemical composition was TiO2. The surface roughness had an average height deviation of 0.83 microm (Sa). The present results are needed to elucidate the influence of the oxide properties on the biological reaction. The results of animal studies using the presently characterized surface oxides on titanium implants will be published separately.

The global pandemic of coronavirus disease 2019 (COVID-19) is associated with the development of acute respiratory distress syndrome (ARDS), which requires ventilation in critically ill patients. The pathophysiology of ARDS results from acute inflammation within the alveolar space and prevention of normal gas exchange. The increase in proinflammatory cytokines within the lung leads to recruitment of leukocytes, further propagating the local inflammatory response. A consistent finding in ARDS is the deposition of fibrin in the air spaces and lung parenchyma. COVID-19 patients show elevated D-Dimers and fibrinogen. Fibrin deposits are found in the lungs of patients due to the dysregulation of the coagulation and fibrinolytic systems. Tissue factor (TF) is exposed on damaged alveolar endothelial cells and on the surface of leukocytes promoting fibrin deposition, while significantly elevated levels of plasminogen activator inhibitor 1 (PAI-1) from lung epithelium and endothelial cells create a hypofibrinolytic state. Prophylaxis treatment of COVID-19 patients with low molecular weight heparin (LMWH) is important to limit coagulopathy. However, to degrade pre-existing fibrin in the lung it is essential to promote local fibrinolysis. In this review, we discuss the repurposing of fibrinolytic drugs, namely tissue-type plasminogen activator (tPA), to treat COVID-19 associated ARDS. tPA is an approved intravenous thrombolytic treatment, and the nebulizer form has been shown to be effective in plastic bronchitis and is currently in Phase II clinical trial. Nebulizer plasminogen activators may provide a targeted approach in COVID-19 patients to degrade fibrin and improving oxygenation in critically ill patients.